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Multifactorial analysis of conditional reprogramming of human keratinocytes.

Ligaba SB, Khurana A, Graham G, Krawczyk E, Jablonski S, Petricoin EF, Glazer RI, Upadhyay G - PLoS ONE (2015)

Bottom Line: Co-culture of human primary epithelial cells with irradiated 3T3 fibroblast feeder cells (J2 cells) and the Rho kinase inhibitor Y-27632 (Y) allows for the unrestricted growth of cells of epithelial origin by the process termed conditional reprogramming.To better understand the nature of the signaling processes associated with conditionally reprogrammed cells, the effect of the two critical components of the co-culture conditions, J2 cells and Y, on the growth of human foreskin keratinocytes (HFKs) was evaluated by gene expression profiling, reverse-phase protein arrays and siRNA screening.These findings establish a mechanistic basis for the unlimited growth potential of human epithelial cells that will be invaluable to assess the effect of genetic changes in pathologic tissues and their response to therapeutic agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Georgetown University, Washington, DC, 20007, United States of America.

ABSTRACT
Co-culture of human primary epithelial cells with irradiated 3T3 fibroblast feeder cells (J2 cells) and the Rho kinase inhibitor Y-27632 (Y) allows for the unrestricted growth of cells of epithelial origin by the process termed conditional reprogramming. To better understand the nature of the signaling processes associated with conditionally reprogrammed cells, the effect of the two critical components of the co-culture conditions, J2 cells and Y, on the growth of human foreskin keratinocytes (HFKs) was evaluated by gene expression profiling, reverse-phase protein arrays and siRNA screening. J2 cells and Y acted cooperatively to down-regulate differentiation, and upregulate proliferation and cell adhesion, including increased pT308Akt and pERK, and reduced TGF-β pathway signaling. These findings establish a mechanistic basis for the unlimited growth potential of human epithelial cells that will be invaluable to assess the effect of genetic changes in pathologic tissues and their response to therapeutic agents.

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Cell cycle transition of conditionally reprogrammed HFKs.A, HFKs at passage 10 were grown in the presence of either Y-27632 (Y), J2 cells (J2) or J2 cells and Y (J2+Y). Conditional reprograming of HFKs increased G2-M transition. FACS indicated that the combination of J2 cells and Y (J2+Y) increased the percentage of cells in G2-M to 32.3%. B, Co-culture of HFKs with J2 cells maintained the level of cell cycle regulators. J2 cells up-regulated cyclins A and E, MCM4 and pCDK1, as well as the stem cell marker p63 vs. Y alone, and reduced expression of the squamous epithelial marker Involucrin.
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pone.0116755.g001: Cell cycle transition of conditionally reprogrammed HFKs.A, HFKs at passage 10 were grown in the presence of either Y-27632 (Y), J2 cells (J2) or J2 cells and Y (J2+Y). Conditional reprograming of HFKs increased G2-M transition. FACS indicated that the combination of J2 cells and Y (J2+Y) increased the percentage of cells in G2-M to 32.3%. B, Co-culture of HFKs with J2 cells maintained the level of cell cycle regulators. J2 cells up-regulated cyclins A and E, MCM4 and pCDK1, as well as the stem cell marker p63 vs. Y alone, and reduced expression of the squamous epithelial marker Involucrin.

Mentions: The Rho kinase inhibitor Y-27632 has been reported to reduce stress fibers and the actin microarchitecture [15, 16], however cells in coculture do not depict its negative effect on cell growth. Cell cycle analysis showed that HFKs grown in the presence of Y exhibited an increase in S phase transition, whereas co-culture with J2 cells increased G2-M, an effect that was further enhanced in the presence of Y (Fig. 1A). The latter conditions maintained the levels of pRb, cyclin A, cyclin E, MCM4 and pCDK1 (Fig. 1B, Fig. B in S1 File). Additionally, J2 cells and Y increased expression of the stem cell marker, p63, and reduced expression of the stratified squamous epithelial cell marker, Involucrin (Fig. 1B, Fig. B in S1 File). Conditional reprogramming did not result in major changes in apoptosis as detected by Annexin V expression and the absence of caspase9 and caspase3 cleavage (Fig. C. I in S1 File). Autophagy was reduced by J2 cells as shown by the absence of cleavage of LC3B, an ubiquitin-like modifier involved in formation of autophagosome vacuoles (Fig. C. II in S1 File).


Multifactorial analysis of conditional reprogramming of human keratinocytes.

Ligaba SB, Khurana A, Graham G, Krawczyk E, Jablonski S, Petricoin EF, Glazer RI, Upadhyay G - PLoS ONE (2015)

Cell cycle transition of conditionally reprogrammed HFKs.A, HFKs at passage 10 were grown in the presence of either Y-27632 (Y), J2 cells (J2) or J2 cells and Y (J2+Y). Conditional reprograming of HFKs increased G2-M transition. FACS indicated that the combination of J2 cells and Y (J2+Y) increased the percentage of cells in G2-M to 32.3%. B, Co-culture of HFKs with J2 cells maintained the level of cell cycle regulators. J2 cells up-regulated cyclins A and E, MCM4 and pCDK1, as well as the stem cell marker p63 vs. Y alone, and reduced expression of the squamous epithelial marker Involucrin.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4340869&req=5

pone.0116755.g001: Cell cycle transition of conditionally reprogrammed HFKs.A, HFKs at passage 10 were grown in the presence of either Y-27632 (Y), J2 cells (J2) or J2 cells and Y (J2+Y). Conditional reprograming of HFKs increased G2-M transition. FACS indicated that the combination of J2 cells and Y (J2+Y) increased the percentage of cells in G2-M to 32.3%. B, Co-culture of HFKs with J2 cells maintained the level of cell cycle regulators. J2 cells up-regulated cyclins A and E, MCM4 and pCDK1, as well as the stem cell marker p63 vs. Y alone, and reduced expression of the squamous epithelial marker Involucrin.
Mentions: The Rho kinase inhibitor Y-27632 has been reported to reduce stress fibers and the actin microarchitecture [15, 16], however cells in coculture do not depict its negative effect on cell growth. Cell cycle analysis showed that HFKs grown in the presence of Y exhibited an increase in S phase transition, whereas co-culture with J2 cells increased G2-M, an effect that was further enhanced in the presence of Y (Fig. 1A). The latter conditions maintained the levels of pRb, cyclin A, cyclin E, MCM4 and pCDK1 (Fig. 1B, Fig. B in S1 File). Additionally, J2 cells and Y increased expression of the stem cell marker, p63, and reduced expression of the stratified squamous epithelial cell marker, Involucrin (Fig. 1B, Fig. B in S1 File). Conditional reprogramming did not result in major changes in apoptosis as detected by Annexin V expression and the absence of caspase9 and caspase3 cleavage (Fig. C. I in S1 File). Autophagy was reduced by J2 cells as shown by the absence of cleavage of LC3B, an ubiquitin-like modifier involved in formation of autophagosome vacuoles (Fig. C. II in S1 File).

Bottom Line: Co-culture of human primary epithelial cells with irradiated 3T3 fibroblast feeder cells (J2 cells) and the Rho kinase inhibitor Y-27632 (Y) allows for the unrestricted growth of cells of epithelial origin by the process termed conditional reprogramming.To better understand the nature of the signaling processes associated with conditionally reprogrammed cells, the effect of the two critical components of the co-culture conditions, J2 cells and Y, on the growth of human foreskin keratinocytes (HFKs) was evaluated by gene expression profiling, reverse-phase protein arrays and siRNA screening.These findings establish a mechanistic basis for the unlimited growth potential of human epithelial cells that will be invaluable to assess the effect of genetic changes in pathologic tissues and their response to therapeutic agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Georgetown University, Washington, DC, 20007, United States of America.

ABSTRACT
Co-culture of human primary epithelial cells with irradiated 3T3 fibroblast feeder cells (J2 cells) and the Rho kinase inhibitor Y-27632 (Y) allows for the unrestricted growth of cells of epithelial origin by the process termed conditional reprogramming. To better understand the nature of the signaling processes associated with conditionally reprogrammed cells, the effect of the two critical components of the co-culture conditions, J2 cells and Y, on the growth of human foreskin keratinocytes (HFKs) was evaluated by gene expression profiling, reverse-phase protein arrays and siRNA screening. J2 cells and Y acted cooperatively to down-regulate differentiation, and upregulate proliferation and cell adhesion, including increased pT308Akt and pERK, and reduced TGF-β pathway signaling. These findings establish a mechanistic basis for the unlimited growth potential of human epithelial cells that will be invaluable to assess the effect of genetic changes in pathologic tissues and their response to therapeutic agents.

Show MeSH
Related in: MedlinePlus